reduced polyglutamine protein aggregation in the primary
human fibroblast cell lines (Figure 7C). In addition, triglyceride
levels after PHX treatment were increased (Figure S6B). Simi-
A
B
Filter Trap Assay
Control(EV) hsp-6 RNAi DMSO PHX
Total Protein
Q35::YFP
Tubulin
Q35::YFP
Control(EV) hsp-6 RNAi DMSO PHX
unc-54p::Q35::YFP
020406080100120day 1 day 2 day 3 day 4Body bends (30sec)
Control (EV)
hsp-6 RNAi**
**
n.s
Days after RNAi0204060801001201401601800 1 2 3 4 5 6 7 8 9 10Number of YFP puncta
Days after RNAiControl (EV)
hsp-6 RNAi
daf-2 RNAiunc-54p::Q35::YFP
EV (day 3) hsp-6 (day3)HeadTailunc-54p::Q35::YFP
Figure 6. Cytosolic Stress Response after
Mitochondrial HSP70 Knockdown Improved
Cytosolic Protein Homeostasis in polyQ-
ExpressingC.elegans
(A) (Top) AM140 worms expressing polyQ
(Q35::YFP) in body wall muscle cells were tested
for motility assay after RNAi treatment. Number of
body bends was measured for 30 s in M9 buffer
(mean±SD of three biological repeats; **p%0.01,
****p%0.0001). (Bottom) The number of YFP
puncta of AM140 worms is shown over time. The
images of YFP puncta in the head and tail showed
more aggregated YFP puncta in control worms
and more soluble YFP signaling inhsp-6RNAi-
treated worms.
(B)hsp-6RNAi- or PHX-treated AM140 worms
showed fewer aggregates via filter trap assay.larly, the mRNA levels of many lipid syn-
thesis genes were increased after
mtHSP70 knockdown, consistent with
our findings in C. elegans (Fig-
ure S6C). Moreover, double knockdown
of mtHSP70 with lipid synthesis enzymes
(acc1orfas) compromised proteostasis
and the cells were no longer able to
reduce polyQ aggregates, consistent
with theC. elegansfindings (Figure S6D).
These results indicate that the MCSR
and its protective management of polyQ
aggregates are conserved from
C. elegansto human cells.DISCUSSIONWe find that mitochondrial perturbation
caused by knocking down mtHSP70 in-
duces a distinct stress response that not
only turns on the UPRmtbut also activates
the HSR and is co-regulated by both
dve-1andhsf-1. We have termed this
stress response the MCSR (Figure 7D).
In summary, our results indicate that
mtHSP70 knockdown or CPT inhibition
can send a retrograde signal to the nu-
cleus to turn on the MCSR to improve
protein homeostasis in both the mito-
chondria and cytosol (Figure 7D). We
thus propose a model in which ceramide
serves as an inhibitor of MCSR under
non-stressed conditions. In the presence
of stress, such as the reduction of
mtHSP70 expression or possibly other
forms of severe mitochondrial dysfunc-
tion, cardiolipins and other lipids accumu-
late, acting as inhibitors of ceramide synthesis and shifting the
metabolic state of the cell, thereby promoting the induction of
the MCSR. Sincehsp-16.2reporter induction by cardiolipin1548 Cell 166 , 1539–1552, September 8, 2016